Figure 1: Clathrin-mediated endocytosis of VEGFR2 is consistently inhibited by either knockdown of clathrin or by treatment with dynasore.

(a) Serum starved (2 h) HUVECs, that were transfected with siRNAs against CHC, were incubated with a mouse anti-VEGFR2 extracellular domain antibody at 4 °C, transferred to 37 °C and stimulated with VEGF, in the presence of FITC-transferrin. Prior to fixation, membrane bound antibodies and transferrin were removed by acid wash and the internalised receptor was revealed by secondary fluorescent antibodies, using confocal microscopy. Nuclei are shown in blue (10 μm scale bars). Quantification of VEGFR2 internalisation and transferrin is shown at the bottom of the immunofluorescence images (20 cells from 3 independent experiments were analysed, mean ± S.D., ***P < 0.001, t-test). (b) 2 h serum starved HUVECs were incubated with a mouse anti-VEGFR2 extracellular domain antibody at 4 °C, treated with vehicle (control) or dynasore at 4 °C for 30 min, transferred to 37 °C and processed as described in “a”. Nuclei are shown in blue (10 μm scale bars). Quantification of VEGFR2 internalisation and transferrin is shown at the bottom of the immunofluorescence images (20 cells from 3 independent experiments were analysed, mean ± S.D., *P < 0.05, ***P < 0.001, t-test).